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Cation induced differential effect on structural and functional properties of Mycobacterium tuberculosis alpha-isopropylmalate synthase.

Singh K, Bhakuni V - BMC Struct. Biol. (2007)

Bottom Line: The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure.This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow, India. ksinghcdri@gmail.com <ksinghcdri@gmail.com>

ABSTRACT

Background: Alpha-isopropylmalate synthase (MtalphaIPMS), an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of Mycobacterium tuberculosis is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtalphaIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented.

Results: Effect of cations on the structure and function of the MtalphaIPMS has been studied in detail. The studies for the first time demonstrate that different cations interact specifically at different sites in the enzyme and modulate the enzyme structure differentially. The inhibitors Zn2+ and Cd2+ ions interact directly with the catalytic domain of the enzyme and induce unfolding/denaturation of the domain. The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure. Studies with isolated catalytic TIM barrel domain showed that it can carry out the catalytic function on its own but probably requires the non-catalytic C-terminal domain for optimum functioning. An important observation was that divalent cations induce significant interaction between the regulatory and the catalytic domain of MtalphaIPMS thus inducing structural cooperativity in the enzyme. This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.

Conclusion: The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

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Related in: MedlinePlus

Thermal unfolding of salt-treated MtαIPMS. Effect of increasing temperature on the CD ellipticity at 222 nm of MtαIPMS (profile 1), MtαIMPS in presence of 5 mM KCl (profile 2), 5 mM MgCl2 (profile 3), 0.25 mM ZnCl2 (profile 4) and 0.25 mM CdCl2 (profile 5), respectively. The values have been represented as percentage with the value obtained at 25°C for each sample taken as 100 percent, respectively.
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Figure 8: Thermal unfolding of salt-treated MtαIPMS. Effect of increasing temperature on the CD ellipticity at 222 nm of MtαIPMS (profile 1), MtαIMPS in presence of 5 mM KCl (profile 2), 5 mM MgCl2 (profile 3), 0.25 mM ZnCl2 (profile 4) and 0.25 mM CdCl2 (profile 5), respectively. The values have been represented as percentage with the value obtained at 25°C for each sample taken as 100 percent, respectively.

Mentions: To analyze whether the two structural domains, N-terminal or TIM barrel domain and the C-terminal or regulatory domain, of MtαIPMS interact strongly with each other or they are independent folding/unfolding units of the protein, thermal denaturation studies were carried out by monitoring the loss of secondary structure of enzyme at increasing temperatures. Figure 8 shows the changes in CD ellipticity at 222 nm for MtαIPMS as a function of temperature. A broad sigmoidal transition with an apparent Tm of about 49°C was observed. An interesting observation was that a loss of only about 35% CD ellipticity at 222 nm was associated with the thermal denaturation of MtαIPMS, demonstrating that the major part of the protein molecule is resistant to thermal unfolding. This indicates that MtαIPMS is composed of two unfolding units that behave independently and have different thermal stabilities: one being sensitive to thermal denaturation and the other resistant to it. A similar sensitivity of structural domains to thermal denaturation has been observed for other proteins [13]. Hence, full-length MtαIPMS is structurally a non-cooperative molecule having two distinct structural units that fold/unfold independently of each other.


Cation induced differential effect on structural and functional properties of Mycobacterium tuberculosis alpha-isopropylmalate synthase.

Singh K, Bhakuni V - BMC Struct. Biol. (2007)

Thermal unfolding of salt-treated MtαIPMS. Effect of increasing temperature on the CD ellipticity at 222 nm of MtαIPMS (profile 1), MtαIMPS in presence of 5 mM KCl (profile 2), 5 mM MgCl2 (profile 3), 0.25 mM ZnCl2 (profile 4) and 0.25 mM CdCl2 (profile 5), respectively. The values have been represented as percentage with the value obtained at 25°C for each sample taken as 100 percent, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919377&req=5

Figure 8: Thermal unfolding of salt-treated MtαIPMS. Effect of increasing temperature on the CD ellipticity at 222 nm of MtαIPMS (profile 1), MtαIMPS in presence of 5 mM KCl (profile 2), 5 mM MgCl2 (profile 3), 0.25 mM ZnCl2 (profile 4) and 0.25 mM CdCl2 (profile 5), respectively. The values have been represented as percentage with the value obtained at 25°C for each sample taken as 100 percent, respectively.
Mentions: To analyze whether the two structural domains, N-terminal or TIM barrel domain and the C-terminal or regulatory domain, of MtαIPMS interact strongly with each other or they are independent folding/unfolding units of the protein, thermal denaturation studies were carried out by monitoring the loss of secondary structure of enzyme at increasing temperatures. Figure 8 shows the changes in CD ellipticity at 222 nm for MtαIPMS as a function of temperature. A broad sigmoidal transition with an apparent Tm of about 49°C was observed. An interesting observation was that a loss of only about 35% CD ellipticity at 222 nm was associated with the thermal denaturation of MtαIPMS, demonstrating that the major part of the protein molecule is resistant to thermal unfolding. This indicates that MtαIPMS is composed of two unfolding units that behave independently and have different thermal stabilities: one being sensitive to thermal denaturation and the other resistant to it. A similar sensitivity of structural domains to thermal denaturation has been observed for other proteins [13]. Hence, full-length MtαIPMS is structurally a non-cooperative molecule having two distinct structural units that fold/unfold independently of each other.

Bottom Line: The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure.This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow, India. ksinghcdri@gmail.com <ksinghcdri@gmail.com>

ABSTRACT

Background: Alpha-isopropylmalate synthase (MtalphaIPMS), an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of Mycobacterium tuberculosis is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtalphaIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented.

Results: Effect of cations on the structure and function of the MtalphaIPMS has been studied in detail. The studies for the first time demonstrate that different cations interact specifically at different sites in the enzyme and modulate the enzyme structure differentially. The inhibitors Zn2+ and Cd2+ ions interact directly with the catalytic domain of the enzyme and induce unfolding/denaturation of the domain. The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure. Studies with isolated catalytic TIM barrel domain showed that it can carry out the catalytic function on its own but probably requires the non-catalytic C-terminal domain for optimum functioning. An important observation was that divalent cations induce significant interaction between the regulatory and the catalytic domain of MtalphaIPMS thus inducing structural cooperativity in the enzyme. This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.

Conclusion: The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

Show MeSH
Related in: MedlinePlus